The efficient delivery of biologically active compounds to the intracellular space of cells has been accomplished by the use of a wide variety of vesicles. One particular type of vesicle, liposomes, is one of the most developed types of vesicles for drug delivery. Liposomes are microscopic vesicles that comprise amphipathic molecules that contain both hydrophobic and hydrophilic regions. Liposomes can contain an aqueous volume that is entirely enclosed by a membrane composed of amphipathic molecules (usually phospholipids).
Liposome drug carriers have been under development since the 1970's. Liposomes are formed from one to several different amphipathic molecules. Several methods have also been developed to complex biologically active compounds with liposomes. A biologically active compound can be entrapped within the internal aqueous phase, within the lipid phase, or complexed to the outside of the liposome.
Liposomes can be divided into three groups based upon their overall size and lamellar structure. Small uni-lamellar vesicles (SUV), which are typically prepared by sonication, are 20 to 30 nm in diameter and contain one single lipid bilayer surrounding the aqueous compartment. Multi-lamellar vesicles (MLV) are prepared by simply mixing amphipathic molecules in an aqueous phase and contain multiple aqueous compartments and bilayers. Large uni-lamellar vesicles (LUV) are most commonly prepared by reverse-phase evaporation. After subsequent pore filtration, LUV's are usually 150 to 200 nm in diameter.
Liposomes can also be classified according to mechanisms by which they attach to a target cell. Gangliosides, polysacharrides and polymers such as polyethylene glycol have been attached to liposomes (termed "Stealth Liposomes") to decrease their non-specific uptake by the reticuloendothelial system in vivo. Antibodies, polysaccharides, sugars, and other ligands have been attached to liposomes to enable the tissue and cell specific delivery of biologically active compounds. Other cellular and viral proteins have also been incorporated into liposomes for targeting purposes and for their fusogenic properties.
Liposomes typically deliver a biologically active compound found within their aqueous space to target cells by fusing with either the plasma membrane or an internal membrane of the cell after endocytosis of the liposome. Fusion of the liposome membrane with the cellular membrane is one of the critical steps in the efficient delivery of substances to the cell. Certain types of liposomes are endocytosed by certain types of cells.
If a liposome is endocytosed by a receptor-mediated pathway, then it enters an endosome. In order for the biologically active compound contained within or associated with the liposome to reach its target sites and receptors, it is essential that the compound escape or be released from the endosome and avoid degradation in the lysosomes.
Knowledge of the phases in which liposomes exist has been used to design liposomes that are more efficient in delivering their contents to cells and fusing with the cellular membranes. Liposomes can exist in a variety of phases. The phases are classified by their lattice type, chain order, and curvature such as: a) micellar, two-dimensional hexagonal (HI), b) inverted micellar (HII), c) two-dimensional oblique (P), d) one dimensional lamellar, bilayer (L.alpha.), e), three-dimensional cubic (Q), and f) three-dimensional crystalline (C). The hydrocarbon chain order is characterized as: a) .alpha., disordered or fluid, b) .beta.-untilted ordered or gel, and c) .beta.', tilted gel. In terms of the curvature, flat bilayer phases have zero curvature. Non-lamellar phases have non-zero curvaturess, type I (normal) phases have positive curvatures in which the interface curves towards the hydrocarbon chains and type II (inverted) phases have negative curvatures in which the interface curves away the hydrocarbon chains. Transitions between the phases can be induced by varying the phospholipid concentration (lyotropic), the temperature (thermotropic), and other conditions such as pH or ionic strength (isothermal).
On the basis of these principles of liposome phases, negatively-charged, pH-sensitive liposomes have been designed to release their contents outside the endosomes by taking advantage of the endosomes' acidification. In studies using specific ligands to carry pH probes into the endocytic pathway, the pH falls to pH 6.5 within 5 minutes of formation of the endocytic vesicles. Maximal acidification as low as pH 4.6 has been reported as the intravesicular pH in macrophages, but the pH may be higher in other cell types. In fibroblasts or epithelioid cells (CV-1), the endosome pH may be approximately 5.5. Several lipid-enveloped viruses such as influenza, vesicular stomatitis virus and Semliki Forest virus microinject their genome into the cytoplasm of the host cell by fusion of their surrounding endosome membrane after endosome acidification. Therefore, liposomes that will destabilize or fuse with the endosome membrane at mildly acidic pH can release their aqueous contents into the cytoplasm.
Liposomes of various compositions can be induced to fuse at a pH below neutral. The threshold can vary from pH 2 for phosphatidylserine-containing LUV's to near pH 7 for SUV's consisting of phosphatidylethanolamine (PE) and palmitoylhomocysteine. A series of PE bilayer stabilizers possessing titratable acidic headgroups have been utilized in the development of pH-sensitive liposomes. All of the negatively-charged, pH sensitive groups have been carboxylic acids as for example palmitoylhomocysteine, oleic acid, palmitic acid, N-succinyldioleoyl-phosphatidylethanolamine, 2,3-seco-5.alpha.-cholestan-2,3-dioic acid, double chain glycerol-based amphiphiles such as N.alpha.(N-oleoyl-2-aminopalmitoyl)histidine (uses the carboxylic acid group in the histidine for pH sensitivity) and N-(N-oleoyl-2-aminopalmitoyl)serine.
Different mechanisms may be operative in proton-induced membrane fusion in the above pH-sensitive, negatively-charged liposomes. These pH-sensitive, negatively-charged liposomes include mixtures of lipids containing a carboxylic acid group and PE (phosphatidylethanoloamine). At high pH, the carboxylic acid group is negatively charged and the increased size of the head group stabilizes the PE-containing liposomes. Liposomes containing only PE at physiologic pH of 4.5-8 are prone to the HII-phase. The PE-rich liposomes which contain second "stabilizing" amphipaths can be stable at pH&gt;pK of the amphiphile. At pH&lt;pK protonation of the amphipath results in an uncharged or reduced-charge species that is unable to stabilize the PE-rich bilayer. The liposomes leak their aqueous contents and form larger structures with the coalescence of membrane components. Many pH-sensitive, PE-rich liposomes have been shown to deliver a variety of membrane-impermeant compounds to various cell types. The mechanism by which cytoplasmic delivery occurs has not been definitely demonstrated. It is not clear whether pH-sensitive liposomes undergo acid-triggered fusion with the lumenal side of the endocytic vesicle membrane or whether the pH-dependent collapse of large numbers of PE-rich liposomes within endocytic vesicles exerts a general detergent-like effect that leads to gross defects in the endosome's membrane.
Negatively-charged, pH-sensitive liposomes have been used to deliver DNA in a functional and target-specific manner in vitro and in vivo. Therefore, further investigation of the delivery mechanism of pH-sensitive liposomes is required. Negatively-charged, pH-sensitive liposomes have also been used to deliver proteins. In addition, negatively-charge liposomes have serious difficulties that include low-transfection efficiency, low encapsulation of DNA, sonication-induced DNA degradation and the requirement to separate the DNA-liposome complexes from "ghost" vesicles.
Various cationic metal ions and polycations have been shown to induce the fusion of negatively-charged liposomes. Polycations such as mellitin, polymixin B, polylysine and synthetic polymers such as polyethylenimine and poly(allylamine) have been shown to induce fusion at neutral pH while polymeric polycations such as polyhistidine and cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI) induce fusion of negatively-charged liposomes at acidic pH. It is generally believed that these polymeric polycations induce fusion of negatively-charged liposomes by increasing their aggregation and presumably inducing lipid phase separation like the divalent cations. The polymeric nature of the cations is an absolute requirement for fusion since the monomeric or oligomeric cations do not induce fusion. While these polycations have been useful for studying liposome fusion they have not been used to deliver biologic substances into cells whether in culture or in the whole organism. In addition the polycations cause hemolysis and/or hemagglutination.
A variety of viral proteins such as F protein of Sendai virus, the HA protein of influenza virus, and the G protein of the vesicular stomatitis virus and toxins such as diptheria toxin and tetanus have also been shown to induce fusion of liposomes at acidic pH. Also, a variety of synthetic peptides such as the GALA peptide and peptides derived from the influenza virus hemagglutinin have also been shown to induce fusion of liposomes at acidic pH. In addition, cellular proteins such as insulin and clathrin induce fusion of negatively-charged liposomes.
In order to circumvent the above difficulties, much more efficient polynucleotide transfer in vitro has been accomplished with the use of positively-charged liposomes that contain cationic lipids. The cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) was the first cationic lipid used for DNA transfections. DOTMA was combined with dioleoylphosphatidylcholine (DOPE) to form liposomes that spontaneously complexed with polynucleotides (DNA and RNA) and enabled relatively efficient transfections. These cationic liposomes are simply mixed with the polynucleotide and then applied to the cells in culture. Complete entrapment of the DNA or RNA molecules occurs because the positively-charged liposomes naturally complex with negatively-charged polynucleotides. DNA has been shown to induce fusion of cationic liposomes containing DOTMA/DOPE. The procedure with the cationic lipids is generally as or more efficient than the commonly-used procedure involving the co-precipitation of calcium phosphate and DNA.
DOTMA/DOPE liposomes have, however, substantial cytotoxicity, particularly in vivo. A variety of cationic lipids have been made in which a diacylglycerol or cholesterol hydrophobic moiety is linked to a cationic headgroup by metabolically degradable ester bond. These have included 1,2-Bis(oleoyloxy)-3-(4'-trimethylammonio)propane (DOTAP), 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) and cholesteryl (4'-trimethylammonio)butanoate (ChoTB). However, there is no evidence of reduced cytotoxicity in comparison of these ester bond-containing cationic lipids as compared to DOTMA. Stearylamine, a cationic lipid has been used in liposomes but it had great cytotoxicity and was not been reported to mediate DNA transfer. Another detergent, cetyltrimethylammonium bromide (CTAB) when combined with DOPE was able to mediate DNA transfection, but it had significant cytotoxicity. A series of cationic, non-pH sensitive lipids that included DORI (1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide), DORIE (1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide), and DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) have been reported and studied. Other non-pH-sensitive, cationic lipids include: O,O'-didodecyl-N-[p-(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylam monium chloride, Lipospermine, DC-Chol (3.beta.[N-(N', N"-dimethylaminoethane)carbonyl]cholesterol), lipopoly(L-lysine), cationic multilamellar liposomes containing N-(.alpha.-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), TransfectACE.TM. (1:2.5 (w:w) ratio of DDAB which is dimethyl dioctadecylammonium bromide and DOPE) (GIBCO BRL) and lipofectAMINE.TM. (3:1 (w:w) ratio of DOSPA which is 2,3-dioleyloxy-N-[20({2,5-bis[(3-aminopropyl)amino]-1-oxypentyl}amino)ethy l]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminium trifluoroacetate and DOPE)(GIBCO BRL).
Lipofectamine mediates the transfection of cells more efficiently than lipofectin (DOPTMA/DOPE) formulations (considered the standard for comparison purposes), but it also has greater cytotoxicity.
All of the cationic lipids mediate transfection only when the cationic lipid/DNA complexes have a positive-to-negative charge ratio of at least one and a half. As a result, serum, which contains negatively-charged components, inhibits transfection to some extent with all the cationic lipid formulations (Lipofectin, LipofectAMINE, LipofectACE, and DOTAP). Chondroitin sulfate type B was a potent inhibitor of transfection with Lipofectin. LipofectAMINE, which is the most efficient cationic lipid transfection reagent, was inhibited the most by serum. Transfection is substantially inhibited if DNA and cationic lipids are mixed in the presence of serum. Cationic lipid liposomes also completely and stochiometrically inhibited the transfer of plasmid DNA into muscle in vivo. Histologic studies showed that the positively-charged DNA/cationic lipid complexes bound to the negatively-charged extracellular matrix and never gained access to the cellular membrane of the muscle cells.